This invention relates to a novel sorbitol dehydrogenase (in the present invention, sorbitol dehydrogenase means an enzyme capable of catalyzing a reaction for converting D-sorbitol to L-sorbose by oxidation; hereinafter to be referred to as SLDH), a gene encoding the same, a method for producing L-sorbose and 2-keto-L-gulonic acid (hereinafter to be referred to as 2KLGA) by gene manipulation using said gene, and an expression system involved in the production thereof. The present invention further relates to a method for producing L-ascorbic acid or a salt thereof utilizing the 2KLGA obtained by the above-mentioned method.
L-sorbose is an important intermediate for the synthesis of L-ascorbic acid (vitamin C) by the Reichstein method (see FIG. 1). When D-sorbitol is chemically oxidized, approximately a half of the product becomes D-sorbose, whereas when D-sorbitol is brought into contact with a microorganism having an SLDH activity, only an L-enantiomer is obtained in a yield of about 95%. Therefore, a fermentation method has been conventionally used for converting D-sorbitol to L-sorbose.
On the other hand, 2KLGA is industrially synthesized by chemically oxidizing L-sorbose. There are known microorganisms that convert L-sorbose into 2KLGA by a two-step enzymatic oxidation reaction by L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH), but the production amount of 2KLGA is low by these methods.
As a method by which to produce 2KLGA more efficiently than before by a fermentation method, there is mentioned a method comprising isolating an SLDH gene, introducing the gene into a microorganism having an SDH or SNDH activity to give a recombinant microorganism capable of synthesizing 2KLGA from D-sorbitol, and bringing the microorganism into contact with D-sorbitol.
Several types of SLDHs have been isolated [Agric. Biol. Chem., 46(1), 135-141 (1982); Biokhimiia, 43(6), 1067-1078 (1978); J. Biol. Chem., 224, 323 (1957); J. Biol. Chem., 226, 301 (1957); J. Bacteriol., 71, 737 (1956)]. The present inventors have already isolated, from a strain belonging to Gluconobacter oxydans, a gene encoding SLDH which is of a membrane-bound type, consists of two large and small subunits and which binds with cytochrome c-like polypeptide and acts (international patent publication No. WO99/20763). However, there is no report on the cloning of a different type of SLDH gene.
It is therefore an object of the present invention to provide a novel SLDH gene useful for the fermentative production of 2KLGA, and to provide a host microorganism transformed with said gene, particularly a transformant obtained by introducing said gene into a host already having SDH and SNDH activity, or a transformant obtained by introducing said gene together with SDH gene and SNDH gene. Another object of the present invention is to provide a method for producing L-sorbose or 2KLGA from D-sorbitol using said microorganism, and to provide a method for producing L-ascorbic acid from 2KLGA obtained by this method. It is yet another object of the present invention to provide a method for producing a recombinant SLDH by culture of a host microorganism transformed with said SLDH gene and a method for producing L-sorbose by an enzyme method using said SLDH.
The present inventors have conducted intensive studies in an attempt to solve the above-mentioned problems and succeeded in cloning a DNA containing a coding region of SLDH from a chromosomal DNA library of a strain belonging to the genus Gluconobacter having said enzyme activity. As a result of the sequencing, the DNA was confirmed to contain a novel SLDH gene completely different from the SLDH gene previously isolated by the present inventors. Moreover, the present inventors transformed Pseudomonas with an expression vector containing the DNA and succeeded in purifying a recombinant SLDH from the culture of said recombinant Pseudomonas. They have also transformed Pseudomonas transformed with an expression vector containing said DNA, with an expression vector containing an SDH gene and an SNDH gene and efficiently converting D-sorbitol to 2KLGA using the culture of this transformant, which resulted in the completion of the present invention.
Accordingly, the present invention provides the following.
(1) An SLDH having the following physicochemical properties:
(a) action: catalyzes the reaction converting D-sorbitol to L-sorbose
(b) molecular weight: about 54 kDa
(c) coenzyme: NAD(P)+ dependent
(d) substrate specificity: specifically oxidizes sorbitol, mannitol and arabitol, but does not act on xylitol, ribitol, inositol and glycerol.
(2) The SLDH of the above-mentioned (1), which is derived from the strain Gluconobacter oxydans G624.
(3) An SLDH which is originated from the same gene as is the SLDH of the above-mentioned (2) in its molecular evolution.
(4) The SLDH of the above-mentioned (3), which is derived from a bacteria belonging to the genus Gluconobacter.
(5) An SLDH which is the following protein (a) or (b):
(a) a protein consisting of an amino acid sequence depicted in Sequence Listing SEQ ID NO:2
(b) a protein consisting of the same amino acid sequence as (a) above, except that one to several amino acids are deleted, substituted, inserted, added or modified, which catalyzes a reaction converting D-sorbitol to L-sorbose.
(6) A DNA encoding the SLDH of any of the above-mentioned (1) to (5).
(7) The DNA of the above-mentioned (6), which is (a) or (b) of the following:
(a) a DNA having a base sequence of base numbers 537-1991 of the base sequence depicted in Sequence Listing SEQ ID NO:1
(b) a DNA capable of hybridizing to the base sequence of the above-mentioned (a) under stringent conditions.
(8) The DNA of the above-mentioned (6) or (7), which is derived from bacteria belonging to the genus Gluconobacter.
(9) A gene encoding a protein having an SLDH activity, which is a DNA capable of hybridizing a DNA having a base sequence of base numbers 537-1991 of the base sequence depicted in Sequence Listing SEQ ID NO:1and a partial DNA thereof.
(10) A protein derived from the genus Gluconobacter, which is encoded by the gene of the above-mentioned (9) and which has an SLDH activity.
(11) A promoter gene comprising the DNA of the following (a) or (b)
(a) a DNA having a base sequence of base numbers 1-536 of the base sequence depicted in Sequence Listing SEQ ID NO:1
(b) a DNA having a base sequence of the above-mentioned xcx9c(a) wherein one to several bases is (are) deleted, substituted, inserted, added or modified, which DNA shows a promoter activity at least in one microorganism.
(12) A recombinant vector comprising a DNA of any of the above-mentioned (6) to (9).
(13) An expression vector comprising a DNA of any of the s above-mentioned (6) to (9).
(14) The expression vector of the above-mentioned (13), further comprising a DNA encoding an SDH and/or a DNA encoding an SNDH.
(15) A transformant obtained by transforming a host cell with an expression vector of the above-mentioned (13) or (14).
(16) The transformant of the above-mentioned (15), which belongs to a genus selected from the group consisting of Escherichia coli, the genus Pseudomonas, the genus Gluconobacter, the genus Acetobacter and the genus Pseudogluconobacter.
(17) The transformant of the above-mentioned (15) or (16), which is capable of converting D-sorbitol to 2-KLGA.
(18) A method for producing a protein having an SLDH activity, which method comprises culturing a host cell transformed with an expression vector of the above-mentioned (13) in a medium and harvesting the SLDH of any of the above-mentioned (1) to (5) or the protein of (10) from the obtained culture.
(19) A method for producing an L-sorbose, which method comprises culturing a host cell transformed with an expression vector of the above-mentioned (13) in a medium and bringing D-sorbitol into contact with the obtained culture or a treated product thereof.
(20) A method for producing 2-KLGA, which method comprises culturing a host cell transformed with an expression vector containing a DNA encoding an SDH and a DNA encoding an SNDH in a medium and bringing the L-sorbose obtained according to the method of the above-mentioned (19) into contact with the obtained culture or a treated product thereof.
(21) A method for producing 2-KLGA, which method comprises culturing the transformant of the above-mentioned (17) in a medium and bringing D-sorbitol into contact with the obtained culture or a treated product thereof.
(22) A method for producing L-ascorbic acid or an alkali metal salt thereof or an alkaline earth metal salt thereof, which method comprises converting 2-KLGA obtained by the method of the above-mentioned (20) or (21) to L-ascorbic acid or an alkali metal salt thereof or an alkaline earth metal salt thereof.
The recombinant cell that expresses the SLDH gene of the present invention can be a useful means for the fermentative production of L-sorbose and 2KLGA. Therefore, the present invention is extremely useful for facilitated and large-scale production of L-ascorbic acid.